CTTNBP2 Controls Synaptic Expression of Zinc-Related Autism-Associated Proteins and Regulates Synapse Formation and Autism-like Behaviors

Abstract

Highlights d Deletion and autism-linked mutation of Cttnbp2 results in autism-like behaviors d Cttnbp2 deficiency impairs dendritic spine formation and neuronal activation in mice d Cttnbp2 deficiency reduces zinc levels and synaptic targeting of zinc-related proteins d Zinc supplementation and D-cycloserine improve the defects of Cttnbp2-deficient mice

Autism-linked mutations of CTTNBP2 reduce social interaction and impair dendritic spine formation via diverse mechanisms

“…The following antibodies and working concentration were used in this study: anti-CTTNBP2 (A5, A7, and 9W, rabbit, homemade, 0.5 μg/ml) [ 5 , 20 ], anti-Myc tag (9B11, Cell Signaling Technology, 1/1000 for staining; 06-549, Millipore, 1 μg/ml), anti-HA tag (3F10, Roche, 0.5 μg/ml), anti-HA tag (Y-11, Santa Cruz Biotechnology, 0.5 μg/ml), anti-GFP (ab13970, Abcam, 0.5 μg/ml), anti-αtubulin (B-5-1-2, Sigma-Aldrich, 1 μg/ml), anti-acetyl tubulin (6-11B-1, Sigma-Aldrich, 1 μg/ml), anti-βactin (AC-74, Sigma-Aldrich, 1/1000), anti-cortactin (H-191, Santa Cruz Biotechnology, 0.5 μg/ml), anti-FOS (#2250, clone 9F6, Cell Signaling Technology, 1/200), anti-mouse HRP (NA931, GE Healthcare, 1/5000), anti-rabbit HRP (NA934, GE Healthcare, 1/5000), anti-chicken Alexa Fluor 488 (A-11039, Invitrogen, 1 μg/ml), anti-mouse Alexa Fluor 555 (A-21424, Invitrogen, 1 μg/ml), anti-rat Alexa Fluor 594 (A-21209, Invitrogen, 1 μg/ml), and anti-rabbit Alexa Fluor 647 (A-21244, Invitrogen, 2 μg/ml).…”

“…Quantification results of dendritic spine density are shown. The quantification results of vector, WT and R533* have been reported previously [ 20 ]. Since those three groups of experiments were performed at the same time as the experiments on the other mutant proteins targeted for this study, we compare all mutants together here.…”

“…Among the various synapse-associated ASD candidate genes, CTTNBP2 was identified as a strong candidate ASD-linked gene based on several human genetic studies [ 7 , 11 , 17 , 18 ]. A study of Cttnbp2 knockout mice has also indicated that CTTNBP2 regulates social interaction, ultrasonic vocalization and hyperactivity [ 20 ], further strengthening the role of CTTNBP2 in ASD. To date, 38 mutations in the CTTNBP2 gene have been identified in patients with ASD ( https://gene.sfari.org/database/human-gene/CTTNBP2#variants-tab ).…”

“…Instead, a protein species of ~ 90 kDa, i.e. the short form, is the only detectable protein product of CTTNBP2 in the brain [ 5 , 14 , 20 , 21 ]. Therefore, mutations within the CTTNBP2 short form, hereafter simply denoted CTTNBP2, are expected to be more relevant to ASD, so we have focused on seven such ASD-linked mutations in this report.…”

“…This role in synaptic targeting is critical for dendritic spine formation and maintenance because both Cttnbp2 knockdown and expression of a CTTNBP2 mutant that cannot interact with cortactin results in reduced dendritic spine density and size [ 5 ]. Apart from its contribution to cytoskeleton dynamics, proteomic analysis has further indicated that CTTNBP2 controls the synaptic expression of more than a hundred proteins, including SHANK2, SHANK3, striatin (STRN), and RAC3 [ 20 ]. These synaptic proteins may also contribute to the function of CTTNBP2 in neurons.…”